EP3037552A1 - Verbesserte verfahren zur nukleinsäuresequenzierung - Google Patents

Verbesserte verfahren zur nukleinsäuresequenzierung Download PDF

Info

Publication number
EP3037552A1
EP3037552A1 EP15197029.0A EP15197029A EP3037552A1 EP 3037552 A1 EP3037552 A1 EP 3037552A1 EP 15197029 A EP15197029 A EP 15197029A EP 3037552 A1 EP3037552 A1 EP 3037552A1
Authority
EP
European Patent Office
Prior art keywords
dna
nucleic acid
rna
strand
transposon
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP15197029.0A
Other languages
English (en)
French (fr)
Other versions
EP3037552B1 (de
Inventor
Niall Anthony Gormley
Louise Fraser
Paula KOKKO-GONZALES
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Illumina Cambridge Ltd
Original Assignee
Illumina Cambridge Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Illumina Cambridge Ltd filed Critical Illumina Cambridge Ltd
Publication of EP3037552A1 publication Critical patent/EP3037552A1/de
Application granted granted Critical
Publication of EP3037552B1 publication Critical patent/EP3037552B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • Sequencing techniques for sequencing nucleic acids including RNA have been developed. Sequencing techniques include, for example, sequencing-by-synthesis. Sequencing-by-synthesis or cycle sequencing can be accomplished by stepwise addition of nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in U.S. Patent No. 7,427,673 ; U.S. Patent No. 7,414,116 ; WO 04/018497 ; WO 91/06678 ; WO 07/123744 ; and U.S. Patent No. 7,057,026 , the disclosures of which are incorporated herein by reference in their entireties. Alternatively, pyrosequencing techniques may be employed.
  • Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand ( Ronaghi et al., (1996) "Real-time DNA sequencing using detection of pyrophosphate release.” Analytical Biochemistry 242(1), 84-9 ; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11 ; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) "A sequencing method based on real-time pyrophosphate.” Science 281(5375), 363 ; U.S. Patent No. 6,210,891 ; U.S. Patent No.
  • released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated is detected via luciferase-produced photons.
  • ATP adenosine triphosphate
  • Sequencing techniques also include sequencing by ligation techniques. Such techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides and are described in U.S. Patent No 6,969,488 ; U.S. Patent No. 6,172,218 ; and U.S. Patent No. 6,306,597 ; the disclosures of which are incorporated herein by reference in their entireties.
  • Other sequencing techniques include, for example, fluorescent in situ sequencing (FISSEQ), and Massively Parallel Signature Sequencing (MPSS).
  • Preparation of DNA samples for sequencing can be relatively straightforward and include using transposition reactions to fragment and add adaptor sequences to the DNA fragments, which simplifies the sample preparation process. See, e.g., International Publication No. WO 2010/048605 , which is incorporated by reference herein in its entirety.
  • current protocols for sequencing RNA samples employ a sample preparation method that converts the RNA in the sample into a double-stranded cDNA format prior to sequencing.
  • preparation of RNA samples for sequencing is more labor intensive.
  • current protocols are less than optimal in their ability to preserve strand-specific information. More specifically, most methods are not able to preserve strand information about the direction of the original single-stranded RNA molecule after being converted into double stranded cDNA.
  • strand-specific information is important for annotation of new genes and for determining gene expression levels.
  • Some methods attempt to preserve strand specific information by ligating adaptors to the ends of single-stranded RNA molecules.
  • the adaptors can have sequences that provide distinguishable information for both ends of the double stranded cDNA generated from the RNA molecules.
  • this method has disadvantages. For example, if the RNA molecules are fragmented, after fragmentation the internal parts of the molecules lose their directional (i.e., strand specific) information.
  • the method includes the steps of providing a transposase and a transposon composition, providing one or more DNA:RNA duplexes immobilized on a support, and contacting the transposase and transposon composition with the one or more DNA:RNA duplexes under conditions wherein the one or more DNA:RNA duplexes and transposon compositions undergo a transposition reaction to produce one or more tagged DNA:RNA duplexes, wherein the transposon composition comprises a double stranded nucleic acid molecule comprising a transferred strand and a non-transferred strand.
  • the method can also be performed for tagging DNA:DNA duplexes that are immobilized on a solid support.
  • the method includes the steps of providing a transposase and a transposon composition, providing one or more DNA:RNA duplexes immobilized on a support, and contacting the transposase and transposon composition with the one or more DNA:RNA duplexes under conditions wherein the one or more DNA:RNA duplexes and transposon composition undergo a transposition reaction to produce one or more tagged DNA:RNA duplexes.
  • the transposon composition comprises a double stranded nucleic acid molecule comprising a transferred strand and a non-transferred strand.
  • the one or more DNA:RNA duplexes are tagged on the 5' end of the RNA strand.
  • the transferred strand comprises a tag to preserve strand information.
  • the transposition reaction results in a 5' tagged RNA strand comprising the transferred strand of the transposon composition and a gap between the 3' end of the DNA strand and the non-transferred strand of the transposon composition.
  • the method further comprises contacting the one or more tagged DNA:RNA duplexes with a nucleic acid modifying enzyme under conditions to extend the 3' end of the DNA stands to copy the RNA strands to their 5' end.
  • the nucleic acid modifying enzyme can displace the non-transferred strand of the transposon composition.
  • FIG. 1 is a schematic showing an exemplary method provided herein.
  • polyA tailed mRNA is captured on a support (e.g., flowcell) via hybridization to a polyT DNA capture probe (or primer) coupled to the surface of the support.
  • the polyT strand is next extended with a reverse transcriptase polymerase to make a double stranded molecule comprising a DNA:RNA duplex.
  • a transposome complex e.g., Tn5 bound with a transposon (e.g., mosaic end (ME) sequence and sequences complementary to surface amplification primers
  • ME mosaic end
  • a strand displacing polymerase e.g., Bst polymerase
  • Bst polymerase can then be used to extend the 3' end of the DNA strand, displacing the 'non-transferred strand' of the transposome and copying the RNA strand to its 5' DNA chimeric end.
  • the double-stranded molecule can then be amplified (e.g., clustered) and sequenced with a sequencing primer partially comprising the ME sequence and the upstream adaptor sequence.
  • the other end of the molecule (the polyT end) can be sequenced with a primer that anneals upstream of the polyT sequence and is extended with natural dATP nucleotides before commencing cycles of SBS chemistry. Paired end sequencing is also enabled by this method.
  • ssDNA When providing ssDNA for sequencing, a similar approach could be utilized.
  • the 3' end of single stranded DNA polynucleotides could be appended with nucleotides by using terminal deoxynucleotidyl transferase (TdT) and any dNTP such as dATP or dTTP.
  • TdT terminal deoxynucleotidyl transferase
  • any dNTP such as dATP or dTTP.
  • Any method for appending a string of nucleotides to the end of a ssDNA molecule could be used.
  • Figure 8 is an example where polyA containing capture probes are immobilized on the support surface and ssDNA-polyT tailed molecules are captured.
  • Any capture sequence including that of the ssDNA end, could be utilized as long as the complementary sequences are provided by the capture probe on the support and the nucleotides on the ssDNA such that hybridization could occur.
  • Extension of the capture probe to create dsDNA by a DNA polymerase to create a DNA:DNA duplex, transpositional ligation of adaptor oligos and strand displacement amplification as previously described could be performed to provide double stranded molecules for cluster formation.
  • the double-stranded molecule could then be amplified (e.g., cluster amplification) and sequenced.
  • RNA total or polyA enriched
  • a single stranded adaptor molecule is ligated to the 3'end of each fragment comprising the complement of the P7 surface bound primer.
  • the fragments are then added to a support (e.g., flowcell) and captured via hybridization.
  • the hybridized RNA molecules are converted to a DNA:RNA duplex with a reverse transcriptase polymerase.
  • a transposome complex comprising a transposase and an adaptor duplex (e.g., transposon) of a ME sequence with a P5 primer sequence can be used to tagment the duplex.
  • the molecules can be amplified and sequenced.
  • a special support e.g., flowcell
  • a standard grafting primer e.g., P5
  • a modified grafting primer e.g., P7
  • a target specific capture probe to its downstream (3') side.
  • An example of a target specific probe is an oligo sequence complementary to a retroviral reverse transcriptase (e.g., HIV polymerase).
  • Purified viral RNA is added to a support, captured via hybridization, copied with reverse transcriptase and tagmented. Sequencing can be achieved with a primer annealed to the tagmented adaptor or at the other end to the capture probe.
  • the special support contains multiple different target specific capture probes to enable simultaneous capture of many different RNA targets.
  • the use of an in vitro transposition reaction to tag the target DNA:DNA or DNA:RNA duplexes to generate tagged DNA:DNA or DNA:RNA duplexes involves a transposase, a transposon sequence composition, and suitable reaction conditions.
  • transposon refers to a double-stranded DNA that contains the nucleotide sequences that are necessary to form the complex with the transposase or integrase enzyme that is functional in an in vitro transposition reaction.
  • a transposon forms a complex or a synaptic complex or a transposome complex.
  • the transposon can also form a transposome composition with a transposase or integrase that recognizes and binds to the transposon sequence, and which complex is capable of inserting or transposing the transposon into target DNA with which it is incubated in an in vitro transposition reaction.
  • a transposon exhibits two complementary sequences consisting of a transferred transposon sequence or transferred strand and a non-transferred transposon sequence, or non transferred strand.
  • one transposon that forms a complex with a hyperactive Tn5 transposase e.g., EZ-Tn5TM Transposase, EPICENTRE Biotechnologies, Madison, WI, USA
  • EZ-Tn5TM Transposase EZ-Tn5TM Transposase, EPICENTRE Biotechnologies, Madison, WI, USA
  • a transposon composition refers to a composition comprising a transposon (i.e., the minimum double-stranded DNA segment that is capable of acting with a transposase to undergo a transposition reaction), optionally including additional sequences.
  • the transposon composition comprises two transposon oligonucleotides containing the transferred transposon oligonucleotide or transferred strand and the non-transferred strand oligonucleotide or non-transferred strand, which, in combination, exhibit the sequences of the transposon.
  • One or both strands can comprise additional sequence.
  • the transposon can include naturally occurring and/or non-naturally occurring nucleotides and natural or non-natural backbone linkages.
  • the transposon can also include one or more moieties attached to the one or more nucleotides making up the transposon.
  • one or both strands of the transposon may be biotinylated or may contain a label, for example a fluorescent label.
  • transferred transposon oligonucleotide and transferred strand are used interchangeably and refer to the transferred portion of both transposons and transposon compositions, i.e., regardless of whether the transposon end is attached to a tag or other sequence or moiety.
  • non-transferred transposon oligonucleotide and non-transferred strand are used interchangeably and refer to the non-transferred portion of both transposons and transposon compositions.
  • the transposon composition comprises or consists of at least one transposon with one or more other nucleotide sequences in addition to the transposon sequences.
  • the transposon composition comprises a transferred strand with one or more other nucleotide sequences 5' of the transferred transposon sequence, e.g., a tag sequence.
  • the tag can have one or more other tag portions or tag domains.
  • a "tag” refers to a nucleic acid component, generally DNA, which provides a means of identifying or addressing a nucleic acid fragment to which it is joined.
  • a tag comprises a nucleotide sequence that permits identification, recognition, and/or molecular or biochemical manipulation of the DNA to which the tag is attached (e.g., by providing a site for annealing an oligonucleotide, such as a primer for extension by a DNA polymerase, by providing an oligonucleotide for capture or for a ligation reaction, or by providing identification of the nucleic acid as originating from a particular source, and the like).
  • tagging The process of joining the tag to a nucleic acid molecule is sometimes referred to herein as "tagging” and nucleic acids that undergoes tagging or that contains a tag is referred to as "tagged” (e.g., "tagged RNA”).”
  • strandedness or strand-specific information refers to the preservation of the knowledge about the direction of the original single-stranded molecule. This is preserved in the provided methods since it is known that the DNA strand is complementary to the RNA strand in the DNA:RNA duplexes. Thus, when sequencing the DNA strand, the sequence will be the sequence of the RNA strand preserving the strand-specific information and allowing for correct identification of the RNA molecule and/or its expression level. Methods for preserving strand specific information are also described in WO 2011/003630 , which is incorporated by reference herein in its entirety.
  • WO 2011/003630 still requires conversion of the RNA molecules into double-stranded cDNA molecules, which, as described herein, is not as efficient as the methods provided in the present application. Further, the method described in WO 2011/003630 requires a tag in order to preserve strand information. In the methods provided herein, a tag is not required to preserve strand-specific information or strandedness.
  • first DNA strand i.e., first DNA strand
  • second DNA strand is the same sequence as the original RNA strand (with the exception of Ts in the sequence instead of Us).
  • a tag e.g., a tag sequence can be included in the transferred strand of the transposon
  • a tag portion or a tag domain means a portion or domain of a tag that exhibits a sequence for a desired intended purpose or application.
  • One tag portion or tag domain is the transposon domain, which tag portion or tag domain exhibits the transferred transposon sequence.
  • the tag also has one or more other tag domains, each of which tag domains is provided for any desired purpose.
  • a transposon composition can comprise (i) a transferred strand that exhibits one or more additional sequences (in addition to the transposon sequence) can comprise a tag domain selected from among one or more of a restriction site tag domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, an address tag domain, and a transcription promoter domain; and (ii) a non-transferred strand that exhibits the non-transferred transposon sequence.
  • tag domain If a description is used for a tag domain, the names and descriptions of different tag domains are for convenience, such as to make it easier to understand and discuss the intended purposes and applications of the different portions or domains of the tag in different embodiments. However, these names and descriptions are not intended to limit the use or applications of the tag or of any of its tag domains in any way. Thus, any particular tag or tag domain can be used for any purpose in addition to, or in place of the intended or primary purpose or application.
  • one tag domain can comprise two or more other tag domains (e.g., a sequencing tag domain can comprise both a capture tag domain and an amplification tag domain) or one tag domain can provide the functions or purposes or applications of two or more different tag domains (e.g., a capture tag domain can also provide the function or purpose of a sequencing tag domain and/or an amplification tag domain for a particular application). Still further, the tag need not be described in terms of one or more different domains in order to be used for any particular purpose or application or function.
  • transposase refers to an enzyme that is capable of forming a functional complex with a transposon-containing composition (e.g., transposons, transposon compositions) and catalyzing insertion or transposition of the transposon-containing composition into the double-stranded target nucleic acid with which it is incubated in an in vitro transposition reaction.
  • a transposase of the provided methods also includes integrases from retrotransposons and retroviruses.
  • Exemplary transposases that can be used in the provided methods include wild-type or mutant forms of Tn5 transposase and MuA transposase.
  • a "transposition reaction” is a reaction wherein one or more transposons are inserted into target nucleic acids at random sites or almost random sites.
  • Essential components in a transposition reaction are a transposase and DNA oligonucleotides that exhibit the nucleotide sequences of a transposon, including the transferred transposon sequence and its complement (i.e., the non-transferred transposon end sequence) as well as other components needed to form a functional transposition or transposome complex.
  • the method of this invention is exemplified by employing a transposition complex formed by a hyperactive Tn5 transposase and a Tn5-type transposon end or by a MuA or HYPERMu transposase and a Mu transposon end comprising R1 and R2 end sequences (See e.g., Goryshin, I. and Reznikoff, W. S., J. Biol. Chem., 273: 7367, 1998 ; and Mizuuchi, K., Cell, 35: 785, 1983 ; Savilahti, H, et al., EMBO J., 14: 4893, 1995 ; which are incorporated by reference herein in their entireties).
  • any transposition system that is capable of inserting a transposon end in a random or in an almost random manner with sufficient efficiency to tag target nucleic acids for its intended purpose can be used in the provided methods.
  • Other examples of known transposition systems that could be used in the provided methods include but are not limited to Staphylococcus aureus Tn552, Ty1, Transposon Tn7, Tn/O and IS 10, Mariner transposase, Tc1, P Element, Tn3, bacterial insertion sequences, retroviruses, and retrotransposon of yeast (See, e.g., whilo O R et al., J. Bacteriol., 183: 2384-8, 2001 ; Kirby C et al., Mol.
  • the method for inserting a transposon into a target sequence can be carried out in vitro using any suitable transposon system for which a suitable in vitro transposition system is available or can be developed based on knowledge in the art.
  • a suitable in vitro transposition system for use in the methods of the present invention requires, at a minimum, a transposase enzyme of sufficient purity, sufficient concentration, and sufficient in vitro transposition activity and a transposon with which the transposase forms a functional complex with the respective transposase that is capable of catalyzing the transposition reaction.
  • transposase transposon sequences that can be used in the invention include but are not limited to wild-type, derivative or mutant transposon sequences that form a complex with a transposase chosen from among a wild-type, derivative or mutant form of the transposase.
  • the DNA:RNA duplexes can be provided in a variety of ways.
  • the support can comprise a plurality of primers and the DNA:RNA duplexes are provided by hybridizing one or more RNA molecules to the immobilized primers on the support and extending the primers hybridized to the RNA molecules using the RNA molecules as template to produce the one or more DNA:RNA duplexes.
  • a plurality of DNA:RNA duplexes are provided by hybridizing a plurality of RNA molecules to the immobilized primers on the support and extending the primers hybridized to the RNA molecules using the RNA molecules as template to produce the plurality of DNA:RNA duplexes.
  • the methods can comprise providing a support with a plurality of primers; the primers or a subset thereof comprising a sequence capable of binding to one or more RNA molecules.
  • the immobilized primers may include a polyT sequence and the RNA may include a polyA sequence capable of hybridizing to the polyT sequence.
  • the plurality of immobilized primers can include target specific primers capable of hybridizing to one or more of the RNA molecules in the plurality of RNA molecules.
  • the RNA strand of the one or more DNA:RNA duplexes comprises a sequence complementary to at least a portion of one or more of the immobilized primers.
  • the plurality of immobilized primers comprises a first subset of primers of a first sequence and a second subset of primers of a second sequence. The first or second subset of primers may comprise a polyT sequence.
  • a 3' adaptor can be added to the plurality of RNA molecules, the 3' adaptor comprising a sequence complementary to the plurality of immobilized primers or a subset thereof.
  • Such 3'-adaptor ligated RNA molecules can then be hybridized to the immobilized primers.
  • the immobilized primers or a subset thereof can comprise a polyT sequence, an RNA target specific sequence or a sequence complementary to an adaptor ligated to the RNA molecule.
  • the plurality of primers comprises at least two subsets of primers, the first subset comprising a polyT sequence, an RNA target specific sequence or a sequence complementary to an adaptor ligated to the RNA molecule, and the second subset of primers comprising a sequence that is capable of binding to a sequence on the DNA strand of the DNA:RNA duplexes.
  • a sequence can be, for example, the same sequence as a sequence of the transferred strand of the transposon.
  • the DNA strand can then be extended to copy the RNA strand.
  • the copying will include copying the sequences of the transferred strand of the transposon.
  • the DNA strand will then include sequences complementary to the sequences of the transferred strand of the transposon and, thus, the primers or subset thereof on the surface of the support.
  • the primers comprises a sequence the same as or similar to the transferred strand of the transposon
  • the DNA strand in the DNA:RNA duplexes will then be capable of hybridizing to the primers since the DNA strand contains a sequence complementary to the primer.
  • Suitable nucleic acid modifying enzymes capable of extending the 3' end of the DNA strands to copy the RNA strands to their 5' end and displacing the non-transferred strand of the transposon are known. Briefly, some DNA polymerases are able to displace the strand complementary to the template strand as a new DNA strand is synthesized by the polymerase. This process is called strand displacement and the DNA polymerases that have this activity are referred to herein as strand-displacing DNA polymerases.
  • a DNA-template-specific DNA polymerase used for the provided methods efficiently synthesizes DNA of a suitable length for the intended purpose without disengaging from the template (or terminating synthesis of the DNA), which is referred to as the enzyme's processivity.
  • the capability of a DNA polymerase to strand displace can be readily determined using the polymerase in a rolling circle replication assay as described by Fire and Xu (Proc. Natl. Acad. Sci. USA 92: 4641-4645, 1995 ), which is incorporated by reference herein in its entirety. Strand displacement and DNA polymerase processivity can also be assayed using methods described in Kong et al. (J. Biol. Chem.
  • Terminal transferase is also defined as a DNA polymerase herein, which DNA polymerase is used as a composition in some embodiments of the provided methods. Terminal transferase can be used because it catalyzes template-independent addition of dNTPs to the 3'-hydroxyl termini of DNA.
  • the method can further comprise sequencing at least a portion of the DNA strands and/or amplifying at least a portion of the DNA strands.
  • the RNA strands from the DNA:RNA duplexes can be removed prior to sequencing and/or amplification.
  • the method further comprises removing the RNA strands from the DNA:RNA duplexes and sequencing at least a portion of the DNA strands (i.e., the first DNA strands).
  • the method can also include copying at least a portion of the DNA strands to produce a second DNA strand complementary to the DNA strand (i.e., the first DNA strand) of the DNA:RNA duplexes.
  • the second complementary DNA strand can then be sequenced, if desired.
  • the first DNA strand of the DNA:RNA duplexes can be removed prior to sequencing the second complementary DNA strand.
  • the DNA strands may be amplified to produce a plurality of double stranded DNA molecules comprising first and second amplified strands.
  • the amplification produces a cluster, described in more detail below.
  • either one or both of the strands can be sequenced.
  • the methods can include removing the first amplified strands followed by sequencing at least a portion of the second amplified strands.
  • the first amplified strands can be regenerated by copying at least a portion of the second amplified strands.
  • the second amplified strands can then be removed in order to sequence at least a portion of the first amplified strands.
  • sequence reads of a portion or all of one or both of the first and second amplified strands can be performed without removing all or a portion of either strand.
  • nucleic acids amplified on a support can be amplified by emulsion PCR, or bridge PCR ( Mitra & Church Nucleic Acids Res. 27, e34 (1999 ); Dressman et al. Proc. Natal. Acad. Sci. USA 100, 8817-8822 (2003 ); Adessi, C. et al. Nucleic Acids Res. 28, e87 (2000 ); Fedurco et al. Nucleic Acids Res. 34, e22 (2006 ), each of which is incorporated herein by reference).
  • nucleic acids can be PCR amplified in a water-in-oil emulsion.
  • a single primer pair is used.
  • One of the PCR primers is tethered to the surface (5'-attached) of a support (e.g., micron-scale beads) and the other primer is in solution.
  • the support comprises primers of more than one sequence, the primers being target specific primers capable of hybridizing to one or more target RNA molecules and the primer in solution is of the same sequence (e.g., a sequence complementary to the sequence added to the DNA strand by copying the tagged RNA strand to its 5' end).
  • RNA molecules can be captured and/or the corresponding DNA complement of the RNA molecule amplified at the surface of the bead. After breaking the emulsion, beads bearing amplification products can be selectively enriched. Each clonally amplified bead will bear on its surface PCR products corresponding to amplification of a single molecule from the template library.
  • emulsion PCR methods that are useful are set forth in U.S. Pat. App. Publ. Nos. 2005/0042648 A1 ; 2005/0079510 A1 and 2005/0130173 A1 , and WO 05/010145 , each of which is incorporated herein by reference.
  • nucleic acids from a template library can be amplified using primers coated on the surface of a support.
  • the primers can be attached at their 5' ends by a flexible linker. Amplification products originating from any given member of the template library remain locally tethered near the point of origin.
  • each clonal cluster contains several copies of a single member of the template library.
  • each DNA:RNA duplex forms the origin of a clonal cluster.
  • the DNA strand can be copied using the primers attached to the support to generate amplified copies of the DNA strand and to produce the clonal cluster.
  • the methods set forth herein can make or use arrays having features at any of a variety of densities including, for example, at least about 10 features/cm 2 , 100 features/cm 2 , 500 features/cm 2 , 1,000 features/cm 2 , 5,000 features/cm 2 , 10,000 features/cm 2 , 50,000 features/cm 2 , 100,000 features/cm 2 , 1,000,000 features/cm 2 , 5,000,000 features/cm 2 , or higher.
  • nucleic acid can be used refer to at least two nucleotide analog monomers linked together.
  • a nucleic acid can contain phosphodiester bonds, however, in some embodiments, a nucleic acid can be an analog having other types of backbones, comprising, for example, phosphoramide, phosphorothioate, phosphorodithioate, peptide nucleic acid backbones and linkages, positive backbones, or non-ionic backbones.
  • a nucleic acid can include a pentose moiety such as ribose (present in naturally occurring RNA), deoxy-ribose (present in naturally occurring DNA) or dideoxy ribose.
  • a nucleic acid can have a non-pentose moiety or carbocyclic sugar instead of a ribose or deoxyribose moiety.
  • a nucleic acid can have one or more different base moieties including, but not limited to, adenine (A), guanine (G), thymine (T), uracil (U), cytosine (C), inosine, xanthanine, hypoxanthanine, isocytosine, isoguanine, nitropyrrole (including 3-nitropyrrole) and/or nitroindole (including 5-nitroindole).
  • a nucleic acid used herein can include native or non-native bases.
  • nucleic acid can include naturally occurring and/or non-naturally occurring nucleotides and natural or non-natural backbone linkages.
  • Nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded and single stranded sequence.
  • the nucleic acid may be DNA (e.g. genomic DNA or cDNA), RNA or a hybrid.
  • an array means a population of different molecules that are attached to one or more supports such that the different molecules can be differentiated from each other according to their relative location.
  • An array can include different molecules that are each located at a different addressable location (e.g. a feature) on a support.
  • an array can include separate supports each bearing a different molecule, wherein the different probe molecules can be identified according to the locations of the supports on a surface to which the supports are attached or according to the locations of the supports in a liquid such as a fluid stream.
  • the molecules of the array can be, for example, nucleic acid primers, nucleic acid probes, nucleic acid templates or nucleic acid enzymes such as polymerases.
  • target nucleic acids can be attached to a surface of a detector or to a layer (e.g. an acrylamide layer) that is present at the surface of the support.
  • Hydrogels are particularly useful such as those set forth in US Pat. Pub. No. 2011/0059865 A1 , which is incorporated herein by reference.
  • array of nucleic acids means a solid support having a plurality of spatially distinguishable nucleic acids disposed thereon or therein.
  • the nucleic acids can be disposed in an ordered or random pattern of features.
  • An individual feature can be, for example, a spatially isolated nucleic acid molecule, or an ensemble of nucleic acid molecules such as a cluster.
  • An array can be a composite array comprising a plurality of individual arrays configured to allow processing of multiple samples.
  • the individual arrays, referred to herein as "sub-arrays,” include groups of nucleic acid features. Sub-arrays appear in distinct regions with in a larger array. The sub-arrays themselves can be ordered or non-ordered.
  • Sub-arrays can be optionally spatially addressable.
  • Sub-arrays can include clusters of identical nucleic acids.
  • An example of a composite array composed of individual sub-arrays is a microtiter plate having wells in which the plate as a whole is an array of nucleic acids (or composite array) while each individual well represents a sub-array within the larger composite array.
  • support refers to a substrate for immobilizing an array of nucleic acids.
  • a “support” is a material having a rigid or semi-rigid surface to which a nucleic acid array can be attached or upon which nucleic acids can be synthesized and/or modified.
  • Supports can include any resin, microbead, glass, controlled pore glass (CPG), polymer support, membrane, paper, plastic, plastic tube or tablet, plastic bead, glass bead, slide, ceramic, silicon chip, multi-well plate, nylon membrane, fiber optic, and PVDF membrane.
  • CPG controlled pore glass
  • a support can include any flat wafer-like substrates and flat substrates having wells, such as a microtiter plate, including 96-well plates.
  • Exemplary flat substrates include chips, slides, etched substrates, microtiter plates, and flow cell reactors, including multi-lane flow cell reactors having multiple microfluidic channels, such as the eight channel flow cell used in the cBot sequencing workstation (Illumina, Inc., San Diego, CA).
  • Exemplary flow cells that can be used are also described in WO 2007/123744 , which is incorporated herein by reference in its entirety.
  • a support can also include beads, including magnetic beads, hollow beads, and solid beads. Beads can be used in conjunction with flat supports, such flat supports optionally also containing wells. Beads, or alternatively microspheres, refer generally to a small body made of a rigid or semi-rigid material. The body can have a shape characterized, for example, as a sphere, oval, microsphere, or other recognized particle shape whether having regular or irregular dimensions.
  • the sizes of beads include, without limitation, about 1 ⁇ m, about 2 ⁇ m, about 3 ⁇ m, about 5 ⁇ m, about 10 ⁇ m, about 20 ⁇ m, about 30 ⁇ m, about 40 ⁇ m, about 60 ⁇ m, about 100 ⁇ m, about 150 ⁇ m or about 200 ⁇ m in diameter.
  • Other particles can be used in ways similar to those described herein for beads and microspheres.
  • composition of a support can vary, depending for example, on the format, chemistry and/or method of attachment and/or on the method of nucleic acid synthesis.
  • Support materials that can be used in accordance with the present disclosure include, but are not limited to, polypropylene, polyethylene, polybutylene, polyurethanes, nylon, metals, and other suitable materials.
  • Exemplary compositions include supports, and chemical functionalities imparted thereto, used in polypeptide, polynucleotide and/or organic moiety synthesis.
  • compositions include, for example, plastics, ceramics, glass, polystyrene, melamine, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex or cross-linked dextrans such as SepharoseTM, cellulose, nylon, cross-linked micelles and TeflonTM, as well as any other materials which can be found described in, for example, "Microsphere Detection Guide " from Bangs Laboratories, Fishers IN, which is incorporated herein by reference.
  • a support particle can be made of cross-linked starch, dextrans, cellulose, proteins, organic polymers including styrene polymers including polystyrene and methylstyrene as well as other styrene co-polymers, plastics, glass, ceramics, acrylic polymers, magnetically responsive materials, colloids, thoriasol, carbon graphite, titanium dioxide, nylon, latex, or TEFLON®.
  • "Microsphere Detection Guide” from Bangs Laboratories, Fishers, Inc. hereby incorporated by reference in its entirety, is a helpful guide.
  • Further exemplary supports within the scope of the present disclosure include, for example, those described in US Application Publication No. 2002/0102578 and U.S. Pat. No. 6,429,027 , both of which are incorporated herein by reference in their entirety.
  • a nucleic acid or other reaction component can be attached to a gel or other semisolid support that is in turn attached or adhered to a solid-phase support. In such embodiments, the nucleic acid or other reaction component will be understood to be solid-phase.
  • the support is a bead or a plurality of beads.
  • the support is a planar support.
  • a plurality of beads is provided, each bead comprising one or more DNA:RNA duplexes. If a bead comprises more than one DNA:RNA duplex, the duplexes can be of the same sequence or different sequence.
  • a plurality of beads is provided each bead comprising a DNA:RNA duplex.
  • the beads in the plurality of beads can comprise the same or a different DNA:RNA duplex.
  • a first subset of beads in the plurality of beads can comprise a DNA:RNA duplex of a first sequence while a second subset of beads in the plurality of beads can comprise a DNA:RNA duplex of a second sequence.
  • Sequencing-by synthesis (SBS) techniques generally involve the enzymatic extension of a nascent nucleic acid strand through the iterative addition of nucleotides against a template strand.
  • SBS can utilize nucleotide monomers that have a terminator moiety or those that lack any terminator moieties.
  • Methods utilizing monomers having terminators include, for example, those described in WO 04/018497 , US 7,057,026 , WO 91/106678 , WO 07/123744 ,U.S.
  • SBS techniques can utilize nucleotide monomers that have a label moiety or those that lack a label moiety. Accordingly, incorporation events can be detected based on a characteristic of the label, such as fluorescence of the label; a characteristic of the nucleotide monomer such as molecular weight or charge; a byproduct of incorporation of the nucleotide, such as release of pyrophosphate or protons; or the like.
  • the different nucleotides can be distinguishable from each other, or alternatively, the two or more different labels can be indistinguishable under the detection techniques being used.
  • the different nucleotides present in a sequencing reagent can have different labels and they can be distinguished using appropriate optics as exemplified by the sequencing methods developed by Solexa (now Illumina, Inc.).
  • Solexa now Illumina, Inc.
  • Methods utilizing nucleotide monomers lacking terminators are also useful including, for example, pyrosequencing.
  • Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand ( Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) "Real-time DNA sequencing using detection of pyrophosphate release.” Analytical Biochemistry 242(1), 84-9 ; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1),3-11 ; Ronaghi, M., Uhlen, M. and Nyren, P.
  • PPi inorganic pyrophosphate
  • Some embodiments can utilize sequencing by ligation techniques. Such techniques utilize DNA ligase to incorporate oligonucleotides. Exemplary SBS systems and methods which can be utilized with the methods and systems described herein are described in U.S. Patent No 6,969,488 , U.S. Patent No. 6,172,218 , and U.S. Patent No. 6,306,597 , the disclosures of which are incorporated herein by reference in their entireties.
  • Some embodiments can utilize methods involving the real-time monitoring of DNA polymerase activity.
  • Nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and y-phosphate-Labeled nucleotides as described, for example, in U.S. Patent No. 7,329,492 and U.S. Patent No. 7,211,414 (each of which is incorporated herein by reference) or nucleotide incorporations can be detected with zero-mode waveguides as described, for example, in U.S. Patent No.
  • FRET fluorescence resonance energy transfer
  • the illumination can be restricted to a zeploliter-scale volume around a surface-tethered polymerase such that incorporation of fluorescently labeled nucleotides can be observed with low background ( Levene, M.1. et al. "Zero-mode waveguides for single-molecule analysis at high concentrations.” Science 299, 682-686 (2003 ); Lundquist, P.M. et al.
  • Example 1 RNA sequencing employing a tagmentation reaction of a DNA:RNA duplex
  • RNA transcripts were generated from a plasmid containing Green Fluorescent Protein (GFP) using the Riboprobe® In vitro Transcription System kit from Promega (Madison, WI) following manufacturer's protocol.
  • GFP Green Fluorescent Protein
  • the sequence of the GFP expression cassette is shown below (SEQ ID NO:3).
  • the pMA-T based plasmid contains P5 (underlined), a T7 polymerase promoter (in bold font), a start codon (in bold font and italics), a His tag (in italics and underlined), a FLAG tag (in capital letters), the GFP sequence, a TAA stop codon (underlined and in bold font), a T7 terminator (in bold font, italics and underlined) and P7' (in capital letters and bold font).
  • the RNA transcript should extend from the promoter sequence to the T7 termination sequence. However, the T7 terminator does not stop transcription completely and so some of the resulting RNA transcripts are His_FLAG_GFP_P7'.
  • the RNA transcript was treated with DNase to remove DNA that would otherwise form clusters. In order to check that the DNase treatment was effective, a reaction was performed and analyzed on a gel to prove that the DNase treatment was effective at removing the DNA. No residual DNA (i.e., plasmid) was visible following DNase treatment of the RNA transcript ( Figure 5 ).
  • a PhiX DNA library and the DNase treated GFP-P7' RNA transcripts were hybridized onto different lanes of a flowcell following the standard cluster protocol for template hybridization.
  • Lanes 1-4 contained the PhiX DNA and lanes 5-8 contained the GFP RNA.
  • Lanes 5 and 6 contained RNA that was pre-treated with DNase to remove DNA.
  • Lanes 7 and 8 contained RNA that was pre-treated with DNase and treated with RNase on the flowcell as an additional control.
  • the PhiX DNA library can hybridize via P5 or P7 as both sequences and their complements are present in the template.
  • the GFP-P7' RNA templates hybridize to the P7 surface primers only because of their 'strandedness' and the lack of a P5 sequence.
  • AMV-RT Avian Myeloblastosis Virus Reverse Transcriptase
  • Phusion DNA polymerase (Lanes 1, 3, 5, and 7).
  • AMV-RT can generate a cDNA strand from either an RNA or DNA template, whereas Phusion can only generate a DNA strand from a DNA template.
  • Some lanes were transposed using a transposome complex containing the transposon sequence P5 adaptor sequence (Lanes 3-8). Gaps in the DNA sequence left after the transposition event were filled in using a strand displacement extension reaction containing Bst DNA polymerase. The transposition event is required in the lanes containing GFP_P7' RNA to add the P5 adapter to generate a template that can make clusters. Isothermal cluster amplification was carried out as standard and the clusters stained with SYBR Green. Pictures of the clusters are shown in Figure 6 .
  • Lane 1 was a control for cluster generation as it contains a standard format DNA sample extended with PHUSION DNA Polymerase. Successful cluster generation resulted as shown in Figure 6 .
  • Lane 2 demonstrated that DNA templates can be successfully extended by a reverse transcriptase (generating a DNA:DNA duplex) and make clusters under standard conditions ( Figure 6 ).
  • Lanes 3 and 4 demonstrated that the DNA:DNA duplexes (extended with either PHUSION DNA Polymerase or AMV-RT) can be tagmented with a Tn5 adaptor and generate clusters ( Figure 6 ).
  • Lane 5 would not be expected to generate clusters because PHUSION DNA Polymerase has been previously reported not to extend opposite an RNA strand. The small number of clusters observed may be due to residual DNA template used to generate the RNA despite DNase treatment, or some degree of extension by PHUSION DNA Polymerase of DNA opposite RNA ( Figure 6 ).
  • Lanes 7 and 8 would not be expected to exhibit any cluster formation because the templates have been RNase and DNase treated. As was seen in lane 5, the small number of clusters observed may be due to residual DNA template used to generate the RNA despite DNase treatment ( Figure 6 ).
  • Lane 6 of Figure 6 demonstrates extension of a DNA strand against an RNA template as expected. These extended templates were not expected to form clusters since they do not possess a P5 sequence. However, following tagmentation with a P5 adaptor ( q.e.d lane 6), they form clusters. The small number of clusters in lanes 5, 7 and 8 suggests there is a low level of DNA contamination in the RNA sample, but shows that the majority of clusters in lane 6 are generated from RNA.
  • Lanes 2, 4, 6, and 8 were amplified with AMV-RT, which amplifies DNA and RNA. Sequencing for non-transposed lanes 1 and 2 with SBS3+T, for transposed lanes 3-8 with Nx R1 primer. Matrix and phasing adjusted, lanes 5-8 aligned to GFP
  • Lanes 3 and 4 which contained tagmented DNA:DNA duplexes exhibited a 10-20% reduction in clusters passing filter, of which between 75-87% of clusters aligned to PhiX. Given that tagmentation can reduce the length of a template, in some cases to a length too short to align effectively, a reduction in cluster passing filters and aligning is not unexpected.
  • the clusters in lanes 7 and 8 should not sequence well since there should not be any template present (with the exception of contaminating DNA templates or undigested RNA stumps).
  • clusters passed filters: less than 7% of clusters passed filters of which only 12% aligned for the PHUSION DNA Polymerase extended templates and 41% aligned for the AMV-RT extended templates. Where no RNase treatment was done, only DNase, and the RNA extended with Phusion 32% of clusters passed filters of which 56+/-16% aligned (Lane 5, Table 1). This may be due to a combination of residual DNA templates and some extension of DNA opposite RNA by PHUSION DNA Polymerase. In contrast, approximately 50% more clusters were observed in lane 6, where DNase treated RNA template was extended with AMV-RT and of which a similar % passed filter ( ⁇ 27%) to lane 6 with 25% aligning.
  • Lane 7 The aligned data was used to generate coverage plots ( Figure 7 ). Lanes 1-4 gave complete genome coverage of PhiX as expected. The lanes containing tagmented DNA (lanes 3 and 4) gave more uneven coverage. Lanes 5-8, containing tagmented RNA samples all showed partial coverage of the GFP, indicating that tagmentation of template has generated clusters. Given that some of this may derive from residual DNA template, lane 6 shows the widest coverage of the GFP template, indicating that AMV-RT extended RNA molecules have been tagmented successfully.
  • Example 2 RNA sequencing employing a tagmentation reaction of human samples.
  • a flowcell with eight lanes was prepared comprising primers capable of hybridizing to RNA molecules comprising a polyA tail as follows.
  • Lane 1 was grafted with a standard oligo mix only comprising P5 and P7 oligos and lanes 2-8 were grafted with standard mix (P5 and P7 oligos) plus the capture oligo (i.e., the primer comprising a polyT sequence for binding to RNA molecules comprising a polyA tail). After primer grafting, the flowcell was stored in 4°C until it was used.
  • RNA sample was prepared and added to the flowcell for hybridization.
  • Lanes 3 and 4 contained human RNA from Clontech (Mountain View, CA).
  • Lanes 5 and 6 contained human RNA from brain.
  • Lanes 7 and 8 contained universal human reference (UHR) RNA.
  • wash buffer was administered through the flowcell for removal of un-hybridized template.
  • Hybridized templates were extended using AMV-RT (NEB, Ipswich, MA) in all lanes, which produced DNA:RNA duplexes in lanes 3-8.
  • transposome complex mixes of two different concentrations were prepared. The mix for lanes 3, 5 and 7 was prepared with 1.25 ⁇ l of transposome complex, 100 ⁇ l of buffer and 400 ⁇ l of water. The mix for lanes 4, 6 and 8 was prepared with 0.625 ⁇ l of transposome complex, 100 ⁇ l of buffer and 400 ⁇ l of water. 95 ⁇ l of transposome complex mixes were added to lanes 3-8 of the flowcell for tagmentation. To remove the transposase after tagmentation, chaotropic buffer was added to lanes 3-8 of the flowcell and incubated for 2 minutes. The lanes of the flowcell were then washed twice.
  • Bst enzyme was used for strand displacement extension of tagmented DNA:RNA duplexes to remove the non-transferred strand of the transposon and make the DNA strand of the DNA:RNA duplexes full length for clustering.
  • the RNA strands were removed and clusters were then generated using isothermal amplification. The clusters were then sequenced. Table 2 shows the results of sequencing. Table 2.
  • the results show normal alignment distribution for the RNA samples sequenced using the tagmentation method provided herein.
  • the results show higher repeat masked clusters likely due to higher numbers of polyA sequences and more repeats in the 3' UTR regions of the RNA samples analyzed by the tagmentation method.
  • the usable reads were about 10% lower than for the standard RNA sequencing protocol again likely due to more repeats in the RNA that was analyzed.
  • the amount of ribosomal RNA is low as would be expected since mRNA was isolated and sequenced in the tagmentation method provided herein.
  • the mitochondrial RNA is within normal limits.
  • RNA sequencing employing a tagmentation reaction and a cell lysate
  • nucleic acid templates can be captured, tagmented and sequenced on a solid support using a crude cell lysate. Briefly, mouse cells were lysed using a Triton-X and Proteinase K solution. The lysate was applied to a flowcell, mRNA was captured and tagmented, and clusters were created and sequenced. As a control and for comparison, Universal Human Reference total RNA (UHR) was also captured, tagmented, clustered and sequenced. Table 4 synopsis the results of duplicate reads for each sample type. Table 4.
  • Table 4 demonstrates that sequence was obtained directly from mRNA captured from a crude mouse cell lysate.
  • the percentage of aligned reads dropped only about 10% when mRNA was captured directly from crude cell lysates compared to the UHR RNA sample (aligned of % PF reads).
  • Further sequencing data comparing the UHR control with mouse lysate derived mRNA reported that the correct strand was captured and aligned at >97% for both the UHR and the mRNA from the mouse lysate. Further, coverage was comparable between the UHR control and the mRNA from lysate; roughly 65% untranslated region (UTR), roughly 16% coding region, roughly 13% intergenic region, and small percentage intronic reads.
  • the present methods can be used to capture, tagment, cluster and sequence mRNA from crude lysates.
  • Example 4 RNA sequencing employing a tagmentation reaction from whole mRNA transcript
  • RNA enrichment was performed from 50ug of UHR total RNA (Agilent) using the PolyA Purist Kit (Ambion).
  • RNA fragmentation of the enriched polyA mRNA was done in 25 ul of 1X T4 PNK Buffer (Epicentre) with 100ng of polyA RNA, wherein the sample was heated to 95°C for 5 min and chilled on ice.
  • the fragmented RNA was phosphorylated with T4 PNK and the fragments were polyA tailed using 4 units of E.
  • coli PolyA polymerase in 50ul of 2X PolyA polymerase buffer containing 2 mM ATP (Epicentre).
  • the polyadenylated fragmented mRNA was purified using the RNA Clean and Concentration kit (Zymo Research). Controls included a PhiX control to validate the sequencing chemistry performance and a non-total mRNA derived polyadenylated sample that was captured and tagmented to compare with the mRNA whole transcript captured from the complex total RNA UHR pool.
  • Table 5 reports that the percentage of aligned reads was comparable regardless of the mRNA source (% align (PF)) with high cluster generation. Additionally, sequence data showed that transcript coverage of the control mRNA (3' capture) was approximately 70% UTR, 19% coding region followed by intergenic and intronic region coverage. Transcript coverage of the mRNA derived from complex total RNA was approximately 43% UTR, 37% coding and relatively similar for intergenic and intronic regions.
  • Figure 10 demonstrates aligned transcript coverage for a representative gene, GAPDH; the control mRNA (3' capture) shows coverage mainly in the 3' region of the gene as expected, whereas coverage from the total RNA derived mRNA (whole transcript) shows more complete coverage of both the exonic and the UTR regions.
  • Double stranded cDNA was prepared from 500ng of UHR total RNA and 50 ng random DNA hexamers. Excess primers were degraded by adding 20 units of Exonuclease I (Epicentre), incubating at 37°C for 30 min followed by enzyme heat inactivation. RNA was removed by an enzyme mix of 1U RNase I/10 U Hybridase (RNAse H, Epicentre) at 55°C for 10 min. The reaction was purified using equal volumes of AMPure beads (Agencourt) and DNA was eluted in a 10mM Tris HCl (pH8.0) buffer.
  • the cDNA was polyA tailed using 20 U Terminal Transferase (New England Biolabs), ImM ATP and 1X Transferase buffer, incubating 37°C for 10 min. followed by heat inactivation. For some of the samples, a 1:10 dilution of the random DNA hexamers was utilized. Further, for some of the samples the Exonuclease I step was omitted. The samples were then applied to a flowcell, captured, tagmented, clustered and sequenced.
  • Controls included PhiX control (ctrl), non tailed cDNA (non tailed ctrl), a dsDNA negative control, and a purified mRNA sample that followed the same method as described above, except using random RNA hexamers and omitting the Exonuclease I step.
  • Table 6 demonstrates that the method of preparation of treating a sample with nucleases, Exo I and RNases H and I, following by bead purification and polyA tailing (Exo, RNAse, AMP, tail) can be used to provide a whole mRNA transcript sample for sequencing.
  • Figure 9 shows pictures of clustering on the flowcell with respect to the different conditions identified in Table 6. Lanes 1-8 in Table 1 correspond to the Lanes 1-8 in Figure 9 .
  • the PhiX positive control shows a large number of clusters which corresponds to the highest yield and cluster count in the sequencing data.
  • the negative control lanes 2 and 8, which show low number of clusters also correspond to two of three the lowest yield and cluster counts in the sequencing data.
  • sample preparation includes, but are not limited to, utilizing the disclosed methods for sequencing RNA from species that do not have polyadenylated RNA, such as bacterial mRNA.
  • ribosomal RNA could first be removed and the remaining mRNA could be fragmented and polyA tailed as previously described. The mRNA could then be captured, tagmented, cluster amplified and sequenced as described above.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
EP15197029.0A 2012-03-06 2013-03-06 Verbesserte verfahren zur nukleinsäuresequenzierung Active EP3037552B1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261607418P 2012-03-06 2012-03-06
EP13714865.6A EP2823058B1 (de) 2012-03-06 2013-03-06 Verbesserte nucleinsäuresequenzierungsverfahren

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
EP13714865.6A Division EP2823058B1 (de) 2012-03-06 2013-03-06 Verbesserte nucleinsäuresequenzierungsverfahren
EP13714865.6A Division-Into EP2823058B1 (de) 2012-03-06 2013-03-06 Verbesserte nucleinsäuresequenzierungsverfahren

Publications (2)

Publication Number Publication Date
EP3037552A1 true EP3037552A1 (de) 2016-06-29
EP3037552B1 EP3037552B1 (de) 2018-04-25

Family

ID=48050665

Family Applications (2)

Application Number Title Priority Date Filing Date
EP15197029.0A Active EP3037552B1 (de) 2012-03-06 2013-03-06 Verbesserte verfahren zur nukleinsäuresequenzierung
EP13714865.6A Active EP2823058B1 (de) 2012-03-06 2013-03-06 Verbesserte nucleinsäuresequenzierungsverfahren

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP13714865.6A Active EP2823058B1 (de) 2012-03-06 2013-03-06 Verbesserte nucleinsäuresequenzierungsverfahren

Country Status (12)

Country Link
US (4) US9574226B2 (de)
EP (2) EP3037552B1 (de)
JP (2) JP6068517B2 (de)
CN (1) CN104160040B (de)
AU (1) AU2013229533B2 (de)
CA (1) CA2864276C (de)
DK (2) DK3037552T5 (de)
ES (1) ES2565809T3 (de)
HK (2) HK1206072A1 (de)
NO (1) NO2694769T3 (de)
TR (1) TR201807138T4 (de)
WO (1) WO2013131962A1 (de)

Families Citing this family (146)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2750879C (en) 2009-01-30 2018-05-22 Oxford Nanopore Technologies Limited Adaptors for nucleic acid constructs in transmembrane sequencing
US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
US20190300945A1 (en) 2010-04-05 2019-10-03 Prognosys Biosciences, Inc. Spatially Encoded Biological Assays
US9074251B2 (en) 2011-02-10 2015-07-07 Illumina, Inc. Linking sequence reads using paired code tags
JP6017458B2 (ja) 2011-02-02 2016-11-02 ユニヴァーシティ・オブ・ワシントン・スルー・イッツ・センター・フォー・コマーシャリゼーション 大量並列連続性マッピング
GB201106254D0 (en) 2011-04-13 2011-05-25 Frisen Jonas Method and product
BR112014001699A2 (pt) 2011-07-25 2017-06-13 Oxford Nanopore Tech Ltd método para sequenciar de um polinucleotídeo alvo de filamento duplo, kit, métodos para preparar um polinucleotídeo alvo de filamento duplo para sequenciamento e para sequenciar um polinucleotídeo alvo de filamento duplo, e, aparelho
EP2875154B1 (de) 2012-07-19 2017-08-23 Oxford Nanopore Technologies Limited SSB-Verfahren zur Charakterisierung einer Nukleinsäure
US9951386B2 (en) 2014-06-26 2018-04-24 10X Genomics, Inc. Methods and systems for processing polynucleotides
CN113528634A (zh) 2012-08-14 2021-10-22 10X基因组学有限公司 微胶囊组合物及方法
US10400280B2 (en) 2012-08-14 2019-09-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11591637B2 (en) 2012-08-14 2023-02-28 10X Genomics, Inc. Compositions and methods for sample processing
US10323279B2 (en) 2012-08-14 2019-06-18 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10273541B2 (en) 2012-08-14 2019-04-30 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10221442B2 (en) 2012-08-14 2019-03-05 10X Genomics, Inc. Compositions and methods for sample processing
US9701998B2 (en) 2012-12-14 2017-07-11 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10752949B2 (en) 2012-08-14 2020-08-25 10X Genomics, Inc. Methods and systems for processing polynucleotides
EP2909337B1 (de) 2012-10-17 2019-01-09 Spatial Transcriptomics AB Verfahren und produkt zur optimierung einer lokalisierten oder räumlichen detektion einer genexpression in einer gewebeprobe
US10533221B2 (en) 2012-12-14 2020-01-14 10X Genomics, Inc. Methods and systems for processing polynucleotides
EP3567116A1 (de) 2012-12-14 2019-11-13 10X Genomics, Inc. Verfahren und systeme zur verarbeitung von polynukleotiden
US9683230B2 (en) 2013-01-09 2017-06-20 Illumina Cambridge Limited Sample preparation on a solid support
KR20200140929A (ko) 2013-02-08 2020-12-16 10엑스 제노믹스, 인크. 폴리뉴클레오티드 바코드 생성
GB201314695D0 (en) 2013-08-16 2013-10-02 Oxford Nanopore Tech Ltd Method
CA2901545C (en) 2013-03-08 2019-10-08 Oxford Nanopore Technologies Limited Use of spacer elements in a nucleic acid to control movement of a helicase
DK3553175T3 (da) 2013-03-13 2021-08-23 Illumina Inc Fremgangsmåde til fremstilling af et nukleinsyresekvenseringsbibliotek
SG11201508985VA (en) 2013-05-23 2015-12-30 Univ Leland Stanford Junior Transposition into native chromatin for personal epigenomics
LT3013983T (lt) 2013-06-25 2023-05-10 Prognosys Biosciences, Inc. Erdviniai koduoti biologiniai tyrimai, naudojant mikrofluidinį įrenginį
AU2014312043A1 (en) 2013-08-30 2016-02-25 Illumina France Manipulation of droplets on hydrophilic or variegated-hydrophilic surfaces
CN114717291A (zh) 2013-12-30 2022-07-08 阿特雷卡公司 使用核酸条形码分析与单细胞缔合的核酸
WO2015106890A1 (en) * 2014-01-14 2015-07-23 Qiagen Gmbh Generation of tagged dna fragments
GB201403096D0 (en) 2014-02-21 2014-04-09 Oxford Nanopore Tech Ltd Sample preparation method
AU2015243445B2 (en) 2014-04-10 2020-05-28 10X Genomics, Inc. Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same
EP3137601B1 (de) 2014-04-29 2020-04-08 Illumina, Inc. Multiplexierte einzelzellengenexpressionsanalyse mittels vorlagenwechsel und tagmentation
WO2015172080A1 (en) 2014-05-08 2015-11-12 Fluidigm Corporation Integrated single cell sequencing
US20150353989A1 (en) 2014-06-09 2015-12-10 Illumina Cambridge Limited Sample preparation for nucleic acid amplification
AU2015273232B2 (en) * 2014-06-13 2021-09-16 Illumina Cambridge Limited Methods and compositions for preparing sequencing libraries
GB201410646D0 (en) * 2014-06-14 2014-07-30 Illumina Cambridge Ltd Methods of increasing sequencing accuracy
CN113249435B (zh) 2014-06-26 2024-09-03 10X基因组学有限公司 分析来自单个细胞或细胞群体的核酸的方法
US10017759B2 (en) * 2014-06-26 2018-07-10 Illumina, Inc. Library preparation of tagged nucleic acid
ES2713153T3 (es) * 2014-06-30 2019-05-20 Illumina Inc Métodos y composiciones que utilizan la transposición unilateral
AU2014406026B2 (en) * 2014-09-12 2018-08-23 Mgi Tech Co., Ltd. Isolated oligonucleotide and use thereof in nucleic acid sequencing
KR20170066540A (ko) 2014-10-09 2017-06-14 일루미나, 인코포레이티드 액체 중 적어도 하나를 효과적으로 격리시키기 위해 비혼화성 액체를 분리하기 위한 방법 및 디바이스
GB201418159D0 (en) 2014-10-14 2014-11-26 Oxford Nanopore Tech Ltd Method
US20170292153A1 (en) * 2014-10-14 2017-10-12 Bgi Shenzhen Co., Limited Method for breaking nucleic acid and adding adaptor by means of transposase, and reagent
KR102643955B1 (ko) * 2014-10-17 2024-03-07 일루미나 케임브리지 리미티드 근접 보존 전위
DK3207134T3 (da) * 2014-10-17 2019-09-23 Illumina Cambridge Ltd Kontiguitetsbevarende transposition
AU2015339148B2 (en) 2014-10-29 2022-03-10 10X Genomics, Inc. Methods and compositions for targeted nucleic acid sequencing
US9975122B2 (en) 2014-11-05 2018-05-22 10X Genomics, Inc. Instrument systems for integrated sample processing
US10006910B2 (en) 2014-12-18 2018-06-26 Agilome, Inc. Chemically-sensitive field effect transistors, systems, and methods for manufacturing and using the same
US9857328B2 (en) 2014-12-18 2018-01-02 Agilome, Inc. Chemically-sensitive field effect transistors, systems and methods for manufacturing and using the same
US9859394B2 (en) 2014-12-18 2018-01-02 Agilome, Inc. Graphene FET devices, systems, and methods of using the same for sequencing nucleic acids
WO2016100049A1 (en) 2014-12-18 2016-06-23 Edico Genome Corporation Chemically-sensitive field effect transistor
US9618474B2 (en) 2014-12-18 2017-04-11 Edico Genome, Inc. Graphene FET devices, systems, and methods of using the same for sequencing nucleic acids
US10020300B2 (en) 2014-12-18 2018-07-10 Agilome, Inc. Graphene FET devices, systems, and methods of using the same for sequencing nucleic acids
WO2016114970A1 (en) 2015-01-12 2016-07-21 10X Genomics, Inc. Processes and systems for preparing nucleic acid sequencing libraries and libraries prepared using same
WO2016138148A1 (en) 2015-02-24 2016-09-01 10X Genomics, Inc. Methods for targeted nucleic acid sequence coverage
US10697000B2 (en) 2015-02-24 2020-06-30 10X Genomics, Inc. Partition processing methods and systems
CN107847930B (zh) 2015-03-20 2020-06-30 亿明达股份有限公司 在竖直或大致竖直的位置中使用的流体盒
EP4321627A3 (de) 2015-04-10 2024-04-17 10x Genomics Sweden AB Räumlich getrennte multiplex-nukleinsäureanalyse von biologischen proben
US11453875B2 (en) 2015-05-28 2022-09-27 Illumina Cambridge Limited Surface-based tagmentation
WO2017007757A1 (en) 2015-07-06 2017-01-12 Illumina, Inc. Balanced ac modulation for driving droplet operations electrodes
CN115369155A (zh) 2015-08-14 2022-11-22 亿明达股份有限公司 使用磁响应式传感器确定遗传特征的系统和方法
JP6743150B2 (ja) 2015-08-28 2020-08-19 イルミナ インコーポレイテッド 単一細胞の核酸配列分析
EP3344389B1 (de) 2015-09-02 2020-06-10 Illumina Cambridge Limited Verfahren zur reparatur von defekten in einer hydrophobischen oberfläche eines tröpfchen-aktuators
US10450598B2 (en) 2015-09-11 2019-10-22 Illumina, Inc. Systems and methods for obtaining a droplet having a designated concentration of a substance-of-interest
US20190217300A1 (en) 2015-10-22 2019-07-18 Illumina, Inc. Filler fluid for fluidic devices
EP3384046B1 (de) 2015-12-01 2021-04-28 Illumina, Inc. Digitales mikrofluidisches system zur einzelligen isolierung und charakterisierung von analyten
WO2017096158A1 (en) 2015-12-04 2017-06-08 10X Genomics, Inc. Methods and compositions for nucleic acid analysis
EP3387152B1 (de) * 2015-12-08 2022-01-26 Twinstrand Biosciences, Inc. Verbesserte adapter, verfahren und zusammensetzungen für duplexsequenzierung
ES2861350T3 (es) 2016-03-28 2021-10-06 Illumina Inc Micromatrices de planos múltiples
CN109312396A (zh) 2016-04-07 2019-02-05 伊鲁米那股份有限公司 用于构建标准化核酸文库的方法和系统
EP4029950A1 (de) 2016-04-29 2022-07-20 Board of Regents, The University of Texas System Gezielte messung der transkriptionellen aktivität im zusammenhang mit hormonrezeptoren
WO2017197338A1 (en) 2016-05-13 2017-11-16 10X Genomics, Inc. Microfluidic systems and methods of use
WO2017201081A1 (en) 2016-05-16 2017-11-23 Agilome, Inc. Graphene fet devices, systems, and methods of using the same for sequencing nucleic acids
GB201609220D0 (en) 2016-05-25 2016-07-06 Oxford Nanopore Tech Ltd Method
CN113151423B (zh) 2016-08-05 2024-09-10 生物辐射实验室股份有限公司 第二链引导
KR102481859B1 (ko) 2016-11-03 2022-12-26 엠쥐아이 테크 컴퍼니 엘티디. 생물학적 또는 화학적 분석을 위한 바이오센서들 및 이를 제조하는 방법
US10815525B2 (en) 2016-12-22 2020-10-27 10X Genomics, Inc. Methods and systems for processing polynucleotides
PT3558510T (pt) * 2016-12-22 2023-01-30 Illumina Inc Matriz que inclui iniciador de sequenciação e entidade não sequenciável
US10011872B1 (en) 2016-12-22 2018-07-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10550429B2 (en) 2016-12-22 2020-02-04 10X Genomics, Inc. Methods and systems for processing polynucleotides
EP4310183A3 (de) 2017-01-30 2024-02-21 10X Genomics, Inc. Verfahren und systeme zur tröpfchenbasierten einzelzellenbarcodierung
DK3452621T3 (da) 2017-02-21 2022-12-12 Illumina Inc Tagmentation ved brug af immobiliserede transposomer med linkere
SG11201908509TA (en) 2017-03-20 2019-10-30 Mgi Tech Co Ltd Biosensors for biological or chemical analysis and methods of manufacturing the same
KR20190133711A (ko) * 2017-03-24 2019-12-03 싱가포르국립대학교 분자의 다중 검출 방법
CN109526228B (zh) 2017-05-26 2022-11-25 10X基因组学有限公司 转座酶可接近性染色质的单细胞分析
US20180340169A1 (en) 2017-05-26 2018-11-29 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
EP3685426A4 (de) 2017-09-19 2021-06-09 MGI Tech Co., Ltd. Herstellung von sequenzierungsdurchflusszellen auf waferebene
WO2019076768A1 (en) 2017-10-16 2019-04-25 Tervisetehnoloogiate Arenduskeskus As METHOD AND KIT FOR PREPARING DNA BANK
WO2019084043A1 (en) 2017-10-26 2019-05-02 10X Genomics, Inc. METHODS AND SYSTEMS FOR NUCLEIC ACID PREPARATION AND CHROMATIN ANALYSIS
CN111051523B (zh) 2017-11-15 2024-03-19 10X基因组学有限公司 功能化凝胶珠
US10829815B2 (en) 2017-11-17 2020-11-10 10X Genomics, Inc. Methods and systems for associating physical and genetic properties of biological particles
KR102313291B1 (ko) * 2017-12-21 2021-10-15 일루미나, 인코포레이티드 하이드로겔 코팅을 가진 플로우 셀
WO2019157529A1 (en) 2018-02-12 2019-08-15 10X Genomics, Inc. Methods characterizing multiple analytes from individual cells or cell populations
WO2019195166A1 (en) 2018-04-06 2019-10-10 10X Genomics, Inc. Systems and methods for quality control in single cell processing
GB201807793D0 (en) 2018-05-14 2018-06-27 Oxford Nanopore Tech Ltd Method
US11932899B2 (en) 2018-06-07 2024-03-19 10X Genomics, Inc. Methods and systems for characterizing nucleic acid molecules
AU2019320771A1 (en) 2018-08-15 2021-02-25 Illumina Cambridge Limited Compositions and methods for improving library enrichment
US20220064630A1 (en) 2018-12-10 2022-03-03 10X Genomics, Inc. Resolving spatial arrays using deconvolution
US11926867B2 (en) 2019-01-06 2024-03-12 10X Genomics, Inc. Generating capture probes for spatial analysis
US11649485B2 (en) 2019-01-06 2023-05-16 10X Genomics, Inc. Generating capture probes for spatial analysis
US11845983B1 (en) 2019-01-09 2023-12-19 10X Genomics, Inc. Methods and systems for multiplexing of droplet based assays
US11467153B2 (en) 2019-02-12 2022-10-11 10X Genomics, Inc. Methods for processing nucleic acid molecules
WO2020168013A1 (en) 2019-02-12 2020-08-20 10X Genomics, Inc. Methods for processing nucleic acid molecules
WO2020177012A1 (zh) * 2019-03-01 2020-09-10 武汉华大医学检验所有限公司 用于rna直接建库的核酸序列、基于rna样本直接构建测序文库的方法及应用
BR112021006173A2 (pt) 2019-04-29 2021-11-16 Illumina Inc Métodos para a identificação e análise de micro-organismos viáveis e/ou proliferativos e para determinação da eficácia de um agente antimicrobiano na modulação do crescimento e da proliferação de micro-organismos em uma amostra
WO2020243579A1 (en) 2019-05-30 2020-12-03 10X Genomics, Inc. Methods of detecting spatial heterogeneity of a biological sample
BR112021012751A2 (pt) * 2019-07-12 2021-12-14 Illumina Cambridge Ltd Preparação de biblioteca de ácidos nucleicos usando eletroforese
GB2604481A (en) 2019-10-10 2022-09-07 1859 Inc Methods and systems for microfluidic screening
WO2021092433A2 (en) 2019-11-08 2021-05-14 10X Genomics, Inc. Enhancing specificity of analyte binding
WO2021091611A1 (en) 2019-11-08 2021-05-14 10X Genomics, Inc. Spatially-tagged analyte capture agents for analyte multiplexing
US20210190770A1 (en) 2019-12-23 2021-06-24 10X Genomics, Inc. Compositions and methods for using fixed biological samples in partition-based assays
ES2982420T3 (es) 2019-12-23 2024-10-16 10X Genomics Inc Métodos para el análisis espacial mediante el uso de la ligazón con plantilla de ARN
US11732299B2 (en) 2020-01-21 2023-08-22 10X Genomics, Inc. Spatial assays with perturbed cells
US11702693B2 (en) 2020-01-21 2023-07-18 10X Genomics, Inc. Methods for printing cells and generating arrays of barcoded cells
US11821035B1 (en) 2020-01-29 2023-11-21 10X Genomics, Inc. Compositions and methods of making gene expression libraries
US12076701B2 (en) 2020-01-31 2024-09-03 10X Genomics, Inc. Capturing oligonucleotides in spatial transcriptomics
US11898205B2 (en) 2020-02-03 2024-02-13 10X Genomics, Inc. Increasing capture efficiency of spatial assays
US12110541B2 (en) 2020-02-03 2024-10-08 10X Genomics, Inc. Methods for preparing high-resolution spatial arrays
US11732300B2 (en) 2020-02-05 2023-08-22 10X Genomics, Inc. Increasing efficiency of spatial analysis in a biological sample
US11835462B2 (en) 2020-02-11 2023-12-05 10X Genomics, Inc. Methods and compositions for partitioning a biological sample
US11891654B2 (en) 2020-02-24 2024-02-06 10X Genomics, Inc. Methods of making gene expression libraries
US11926863B1 (en) 2020-02-27 2024-03-12 10X Genomics, Inc. Solid state single cell method for analyzing fixed biological cells
US11768175B1 (en) 2020-03-04 2023-09-26 10X Genomics, Inc. Electrophoretic methods for spatial analysis
CN115916999A (zh) 2020-04-22 2023-04-04 10X基因组学有限公司 用于使用靶向rna耗竭进行空间分析的方法
EP4153775B1 (de) 2020-05-22 2024-07-24 10X Genomics, Inc. Simultane räumlich-zeitliche messung der genexpression und der zellaktivität
AU2021275906A1 (en) 2020-05-22 2022-12-22 10X Genomics, Inc. Spatial analysis to detect sequence variants
EP4158054A1 (de) 2020-06-02 2023-04-05 10X Genomics, Inc. Räumliche transkriptomik für antigenrezeptoren
US12031177B1 (en) 2020-06-04 2024-07-09 10X Genomics, Inc. Methods of enhancing spatial resolution of transcripts
EP4421186A3 (de) 2020-06-08 2024-09-18 10X Genomics, Inc. Verfahren zur bestimmung eines chirurgischen randes und verfahren zur verwendung davon
WO2021263111A1 (en) 2020-06-25 2021-12-30 10X Genomics, Inc. Spatial analysis of dna methylation
US11761038B1 (en) 2020-07-06 2023-09-19 10X Genomics, Inc. Methods for identifying a location of an RNA in a biological sample
US11981960B1 (en) 2020-07-06 2024-05-14 10X Genomics, Inc. Spatial analysis utilizing degradable hydrogels
CN116171330A (zh) 2020-08-06 2023-05-26 Illumina公司 使用小珠连接的转座体制备rna和dna测序文库
US11981958B1 (en) 2020-08-20 2024-05-14 10X Genomics, Inc. Methods for spatial analysis using DNA capture
US11926822B1 (en) 2020-09-23 2024-03-12 10X Genomics, Inc. Three-dimensional spatial analysis
US11827935B1 (en) 2020-11-19 2023-11-28 10X Genomics, Inc. Methods for spatial analysis using rolling circle amplification and detection probes
EP4121555A1 (de) 2020-12-21 2023-01-25 10X Genomics, Inc. Verfahren, zusammensetzungen und systeme zur erfassung von sonden und/oder barcodes
EP4421491A2 (de) 2021-02-19 2024-08-28 10X Genomics, Inc. Verfahren zur verwendung einer modularen testträgervorrichtung
WO2022198068A1 (en) 2021-03-18 2022-09-22 10X Genomics, Inc. Multiplex capture of gene and protein expression from a biological sample
WO2022256503A1 (en) 2021-06-03 2022-12-08 10X Genomics, Inc. Methods, compositions, kits, and systems for enhancing analyte capture for spatial analysis
WO2022272260A1 (en) 2021-06-23 2022-12-29 Illumina, Inc. Compositions, methods, kits, cartridges, and systems for sequencing reagents
WO2023034489A1 (en) 2021-09-01 2023-03-09 10X Genomics, Inc. Methods, compositions, and kits for blocking a capture probe on a spatial array
WO2023056328A2 (en) * 2021-09-30 2023-04-06 Illumina, Inc. Solid supports and methods for depleting and/or enriching library fragments prepared from biosamples
CN117813397A (zh) 2021-12-21 2024-04-02 因美纳有限公司 蜡-微球基质组合物及其制备和使用方法
WO2023209606A1 (en) 2022-04-29 2023-11-02 Illumina Cambridge Limited Methods and systems for encapsulating lyophilised microspheres

Citations (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006678A1 (en) 1989-10-26 1991-05-16 Sri International Dna sequencing
WO1995023875A1 (en) 1994-03-02 1995-09-08 The Johns Hopkins University In vitro transposition of artificial transposons
US5641658A (en) 1994-08-03 1997-06-24 Mosaic Technologies, Inc. Method for performing amplification of nucleic acid with two primers bound to a single solid support
WO1998044151A1 (en) 1997-04-01 1998-10-08 Glaxo Group Limited Method of nucleic acid amplification
WO2000018957A1 (en) 1998-09-30 2000-04-06 Applied Research Systems Ars Holding N.V. Methods of nucleic acid amplification and sequencing
US6090592A (en) 1994-08-03 2000-07-18 Mosaic Technologies, Inc. Method for performing amplification of nucleic acid on supports
US6172218B1 (en) 1994-10-13 2001-01-09 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
US6274320B1 (en) 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid
US6306597B1 (en) 1995-04-17 2001-10-23 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
WO2002046456A1 (en) 2000-12-08 2002-06-13 Applied Research Systems Ars Holding N.V. Isothermal amplification of nucleic acids on a solid support
US20020102578A1 (en) 2000-02-10 2002-08-01 Todd Dickinson Alternative substrates and formats for bead-based array of arrays TM
US6429027B1 (en) 1998-12-28 2002-08-06 Illumina, Inc. Composite arrays utilizing microspheres
WO2004018497A2 (en) 2002-08-23 2004-03-04 Solexa Limited Modified nucleotides for polynucleotide sequencing
US6737236B1 (en) 1997-01-08 2004-05-18 Proligo, Llc Bioconjugation of macromolecules
WO2005010145A2 (en) 2003-07-05 2005-02-03 The Johns Hopkins University Method and compositions for detection and enumeration of genetic variations
US20050042648A1 (en) 1997-07-07 2005-02-24 Andrew Griffiths Vitro sorting method
US20050079510A1 (en) 2003-01-29 2005-04-14 Jan Berka Bead emulsion nucleic acid amplification
WO2005065814A1 (en) 2004-01-07 2005-07-21 Solexa Limited Modified molecular arrays
US6969488B2 (en) 1998-05-22 2005-11-29 Solexa, Inc. System and apparatus for sequential processing of analytes
US7057026B2 (en) 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
WO2006064199A1 (en) 2004-12-13 2006-06-22 Solexa Limited Improved method of nucleotide detection
US20060240439A1 (en) 2003-09-11 2006-10-26 Smith Geoffrey P Modified polymerases for improved incorporation of nucleotide analogues
US20060281109A1 (en) 2005-05-10 2006-12-14 Barr Ost Tobias W Polymerases
WO2007010251A2 (en) 2005-07-20 2007-01-25 Solexa Limited Preparation of templates for nucleic acid sequencing
US7211414B2 (en) 2000-12-01 2007-05-01 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
US20070128624A1 (en) 2005-11-01 2007-06-07 Gormley Niall A Method of preparing libraries of template polynucleotides
US7259258B2 (en) 2003-12-17 2007-08-21 Illumina, Inc. Methods of attaching biological compounds to solid supports using triazine
WO2007123744A2 (en) 2006-03-31 2007-11-01 Solexa, Inc. Systems and devices for sequence by synthesis analysis
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US7329492B2 (en) 2000-07-07 2008-02-12 Visigen Biotechnologies, Inc. Methods for real-time single molecule sequence determination
US20080108082A1 (en) 2006-10-23 2008-05-08 Pacific Biosciences Of California, Inc. Polymerase enzymes and reagents for enhanced nucleic acid sequencing
US7375234B2 (en) 2002-05-30 2008-05-20 The Scripps Research Institute Copper-catalysed ligation of azides and acetylenes
US7405281B2 (en) 2005-09-29 2008-07-29 Pacific Biosciences Of California, Inc. Fluorescent nucleotide analogs and uses therefor
US7414116B2 (en) 2002-08-23 2008-08-19 Illumina Cambridge Limited Labelled nucleotides
US7427678B2 (en) 1998-01-08 2008-09-23 Sigma-Aldrich Co. Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method
US20090226975A1 (en) 2008-03-10 2009-09-10 Illumina, Inc. Constant cluster seeding
WO2010048605A1 (en) 2008-10-24 2010-04-29 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
WO2011003630A1 (en) 2009-07-06 2011-01-13 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e. V. Method for differentiation of polynucleotide strands

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5523221A (en) * 1993-06-16 1996-06-04 Stratagene Method for the directional cloning of DNA
WO2003093501A2 (en) * 2002-04-30 2003-11-13 University Of Bremen Ssh based methods for identifying and isolating unique nucleic acid sequences
US9080211B2 (en) * 2008-10-24 2015-07-14 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
CA2803693A1 (en) 2010-08-27 2012-03-01 Genentech, Inc. Methods for nucleic acid capture and sequencing
US9029103B2 (en) * 2010-08-27 2015-05-12 Illumina Cambridge Limited Methods for sequencing polynucleotides
ES2568910T3 (es) * 2011-01-28 2016-05-05 Illumina, Inc. Reemplazo de oligonucleótidos para bibliotecas etiquetadas en dos extremos y direccionadas
JP6017458B2 (ja) * 2011-02-02 2016-11-02 ユニヴァーシティ・オブ・ワシントン・スルー・イッツ・センター・フォー・コマーシャリゼーション 大量並列連続性マッピング
DK2691541T3 (en) * 2011-03-31 2018-01-22 Dana Farber Cancer Inst Inc PROCEDURE FOR ENRICHMENT OF SINGLE DRAWED MUTANTS SEQUENCES FROM A MIXTURE OF WILD TYPE AND MUTANTS

Patent Citations (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006678A1 (en) 1989-10-26 1991-05-16 Sri International Dna sequencing
WO1995023875A1 (en) 1994-03-02 1995-09-08 The Johns Hopkins University In vitro transposition of artificial transposons
US6090592A (en) 1994-08-03 2000-07-18 Mosaic Technologies, Inc. Method for performing amplification of nucleic acid on supports
US5641658A (en) 1994-08-03 1997-06-24 Mosaic Technologies, Inc. Method for performing amplification of nucleic acid with two primers bound to a single solid support
US6172218B1 (en) 1994-10-13 2001-01-09 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US6306597B1 (en) 1995-04-17 2001-10-23 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
US6737236B1 (en) 1997-01-08 2004-05-18 Proligo, Llc Bioconjugation of macromolecules
WO1998044151A1 (en) 1997-04-01 1998-10-08 Glaxo Group Limited Method of nucleic acid amplification
US20050100900A1 (en) 1997-04-01 2005-05-12 Manteia Sa Method of nucleic acid amplification
US20050042648A1 (en) 1997-07-07 2005-02-24 Andrew Griffiths Vitro sorting method
US7427678B2 (en) 1998-01-08 2008-09-23 Sigma-Aldrich Co. Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method
US6969488B2 (en) 1998-05-22 2005-11-29 Solexa, Inc. System and apparatus for sequential processing of analytes
WO2000018957A1 (en) 1998-09-30 2000-04-06 Applied Research Systems Ars Holding N.V. Methods of nucleic acid amplification and sequencing
US6429027B1 (en) 1998-12-28 2002-08-06 Illumina, Inc. Composite arrays utilizing microspheres
US6274320B1 (en) 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid
US20020102578A1 (en) 2000-02-10 2002-08-01 Todd Dickinson Alternative substrates and formats for bead-based array of arrays TM
US7329492B2 (en) 2000-07-07 2008-02-12 Visigen Biotechnologies, Inc. Methods for real-time single molecule sequence determination
US7211414B2 (en) 2000-12-01 2007-05-01 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
WO2002046456A1 (en) 2000-12-08 2002-06-13 Applied Research Systems Ars Holding N.V. Isothermal amplification of nucleic acids on a solid support
US20060188901A1 (en) 2001-12-04 2006-08-24 Solexa Limited Labelled nucleotides
US7057026B2 (en) 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
US7427673B2 (en) 2001-12-04 2008-09-23 Illumina Cambridge Limited Labelled nucleotides
US7375234B2 (en) 2002-05-30 2008-05-20 The Scripps Research Institute Copper-catalysed ligation of azides and acetylenes
WO2004018497A2 (en) 2002-08-23 2004-03-04 Solexa Limited Modified nucleotides for polynucleotide sequencing
US20070166705A1 (en) 2002-08-23 2007-07-19 John Milton Modified nucleotides
US7414116B2 (en) 2002-08-23 2008-08-19 Illumina Cambridge Limited Labelled nucleotides
US20050079510A1 (en) 2003-01-29 2005-04-14 Jan Berka Bead emulsion nucleic acid amplification
US20050130173A1 (en) 2003-01-29 2005-06-16 Leamon John H. Methods of amplifying and sequencing nucleic acids
WO2005010145A2 (en) 2003-07-05 2005-02-03 The Johns Hopkins University Method and compositions for detection and enumeration of genetic variations
US20060240439A1 (en) 2003-09-11 2006-10-26 Smith Geoffrey P Modified polymerases for improved incorporation of nucleotide analogues
US7259258B2 (en) 2003-12-17 2007-08-21 Illumina, Inc. Methods of attaching biological compounds to solid supports using triazine
WO2005065814A1 (en) 2004-01-07 2005-07-21 Solexa Limited Modified molecular arrays
US20110059865A1 (en) 2004-01-07 2011-03-10 Mark Edward Brennan Smith Modified Molecular Arrays
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
WO2006064199A1 (en) 2004-12-13 2006-06-22 Solexa Limited Improved method of nucleotide detection
US20060281109A1 (en) 2005-05-10 2006-12-14 Barr Ost Tobias W Polymerases
WO2007010251A2 (en) 2005-07-20 2007-01-25 Solexa Limited Preparation of templates for nucleic acid sequencing
US7405281B2 (en) 2005-09-29 2008-07-29 Pacific Biosciences Of California, Inc. Fluorescent nucleotide analogs and uses therefor
US20070128624A1 (en) 2005-11-01 2007-06-07 Gormley Niall A Method of preparing libraries of template polynucleotides
WO2007123744A2 (en) 2006-03-31 2007-11-01 Solexa, Inc. Systems and devices for sequence by synthesis analysis
US20080108082A1 (en) 2006-10-23 2008-05-08 Pacific Biosciences Of California, Inc. Polymerase enzymes and reagents for enhanced nucleic acid sequencing
US20090226975A1 (en) 2008-03-10 2009-09-10 Illumina, Inc. Constant cluster seeding
WO2010048605A1 (en) 2008-10-24 2010-04-29 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
WO2011003630A1 (en) 2009-07-06 2011-01-13 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e. V. Method for differentiation of polynucleotide strands

Non-Patent Citations (30)

* Cited by examiner, † Cited by third party
Title
ADESSI, C. ET AL., NUCLEIC ACIDS RES., vol. 28, 2000, pages E87
BOEKE J D; CORCES V G, ANNU REV MICROBIOL., vol. 43, 1989, pages 403 - 34
BROWN P 0, PROC NATL ACAD SCI USA, vol. 86, 1989, pages 2525 - 9
CARUCCIO NICHOLAS: "Preparation of next-generation sequencing libraries using Nextera(TM) technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition", METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.) UNITED STATES 2012, TOTOWA, NJ : HUMANA PR., 1984-CLIFTON, NJ : HUMANA PR, US, vol. 733, 1 January 2011 (2011-01-01), pages 241 - 255, XP009157814, ISSN: 1940-6029, [retrieved on 20110101], DOI: 10.1007/978-1-61779-089-8_17 *
COLEGIO 0 R ET AL., J. BACTERIOL., vol. 183, 2001, pages 2384 - 8
CRAIG, N L, CURR TOP MICROBIOL IMMUNOL., vol. 204, 1996, pages 27 - 48
CRAIG, N L, SCIENCE, vol. 271, 1996, pages 1512
DEVINE S E; BOEKE J D., NUCLEIC ACIDS RES., vol. 22, 1994, pages 3765 - 72
DRESSMAN ET AL., PROC. AT . ACAD. SCI. USA, vol. 100, 2003, pages 8817 - 8822
FEDURCO ET AL., NUCLEIC ACIDS RES., vol. 34, 2006
FIRE; XU, PROC. NATL. ACAD. SCI. USA, vol. 92, 1995, pages 4641 - 4645
GLOOR, G B, METHODS MOL. BIOL., vol. 260, 2004, pages 97 - 114
GORYSHIN, I.; REZNIKOFF, W. S., J. BIOL. CHEM., vol. 273, pages 7367
ICHIKAWA H; OHTSUBO E., J BIOL. CHEM., vol. 265, 1990, pages 18829 - 32
KIRBY C ET AL., MOL. MICROBIOL., vol. 43, 2002, pages 173 - 86
KLECKNER N ET AL., CURR TOP MICROBIOL IMMUNOL., vol. 204, 1996, pages 49 - 82
KONG ET AL., J. BIOL. CHEM., vol. 268, 1993, pages 1965 - 1975
KORLACH, J. ET AL.: "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures", PROC. NAT'L. ACAD. SCI. USA, vol. 105, 2008, pages 1176 - 1181
LAMPE D J ET AL., EMBO J., vol. 15, 1996, pages 5470 - 9
LEVENE, M. 1. ET AL.: "Zero-mode waveguides for single-molecule analysis at high concentrations", SCIENCE, vol. 299, 2003, pages 682 - 686
LUNDQUIST, P.M. ET AL.: "Parallel confocal detection of single molecules in real time", OPT. LETT., vol. 33, 2008, pages 1026 - 1028
MITRA; CHURCH, NUCLEIC ACIDS RES., vol. 27, 1999, pages E34
MIZUUCHI, K., CELL, vol. 35, 1983, pages 785
OHTSUBO, F; SEKINE, Y, CURR. TOP. MICROBIOL. IMMUNOL., vol. 204, 1996, pages 1 - 26
PLASTERK R H, CURR TOP MICROBIOL IMMUNOL, vol. 204, 1996, pages 125 - 43
RONAGHI ET AL.: "Real-time DNA sequencing using detection of pyrophosphate release", ANALYTICAL BIOCHEMISTRY, vol. 242, no. I, 1996, pages 84 - 9
RONAGHI, M.: "Pyrosequencing sheds light on DNA sequencing", GENOME RES., vol. 11, no. 1, 2001, pages 3 - 11
RONAGHI, M.; KARAMOHAMED, S.; PETTERSSON, B.; UHLEN, M; NYREN, P.: "Real-time DNA sequencing using detection of pyrophosphate release", ANALYTICAL BIOCHEMISTRY, vol. 242, no. 1, 1996, pages 84 - 9
RONAGHI, M.; UHLEN, M.; NYREN, P.: "A sequencing method based on real-time pyrophosphate", SCIENCE, vol. 281, no. 5375, 1998, pages 363
SAVILAHTI, H ET AL., EMBO J., vol. 14, 1995, pages 4893

Also Published As

Publication number Publication date
TR201807138T4 (tr) 2018-06-21
JP2015509371A (ja) 2015-03-30
CN104160040B (zh) 2017-07-07
JP6341984B2 (ja) 2018-06-13
DK3037552T3 (en) 2018-05-28
US9574226B2 (en) 2017-02-21
AU2013229533A1 (en) 2014-09-04
US20150087534A1 (en) 2015-03-26
ES2565809T3 (es) 2016-04-07
EP2823058A1 (de) 2015-01-14
JP2017079773A (ja) 2017-05-18
CN104160040A (zh) 2014-11-19
CA2864276C (en) 2020-05-05
DK3037552T5 (en) 2018-06-06
US20170212123A1 (en) 2017-07-27
US20240117409A1 (en) 2024-04-11
AU2013229533B2 (en) 2018-06-28
NO2694769T3 (de) 2018-03-03
EP3037552B1 (de) 2018-04-25
WO2013131962A1 (en) 2013-09-12
US11834699B2 (en) 2023-12-05
HK1226105B (zh) 2017-09-22
US10267804B2 (en) 2019-04-23
JP6068517B2 (ja) 2017-01-25
CA2864276A1 (en) 2013-09-12
HK1206072A1 (zh) 2015-12-31
DK2823058T3 (en) 2016-03-21
EP2823058B1 (de) 2016-03-02
US20190324042A1 (en) 2019-10-24

Similar Documents

Publication Publication Date Title
US20240117409A1 (en) Methods of Nucleic Acid Sequencing
JP7203893B2 (ja) 固形支持体でのサンプル調製
EP3981884B1 (de) Einzelzellen-ganzgenombibliotheken zur methylierungssequenzierung
US20240271126A1 (en) Oligo-modified nucleotide analogues for nucleic acid preparation
NZ794511A (en) Single cell whole genome libraries for methylation sequencing

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AC Divisional application: reference to earlier application

Ref document number: 2823058

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20161214

RBV Designated contracting states (corrected)

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20170203

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1226105

Country of ref document: HK

INTG Intention to grant announced

Effective date: 20170830

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAJ Information related to disapproval of communication of intention to grant by the applicant or resumption of examination proceedings by the epo deleted

Free format text: ORIGINAL CODE: EPIDOSDIGR1

GRAL Information related to payment of fee for publishing/printing deleted

Free format text: ORIGINAL CODE: EPIDOSDIGR3

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

GRAR Information related to intention to grant a patent recorded

Free format text: ORIGINAL CODE: EPIDOSNIGR71

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTC Intention to grant announced (deleted)
INTG Intention to grant announced

Effective date: 20180207

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AC Divisional application: reference to earlier application

Ref document number: 2823058

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 992982

Country of ref document: AT

Kind code of ref document: T

Effective date: 20180515

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602013036731

Country of ref document: DE

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

Effective date: 20180525

REG Reference to a national code

Ref country code: NL

Ref legal event code: FP

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: TR-IP CONSULTING LLC, CH

REG Reference to a national code

Ref country code: DK

Ref legal event code: T5

Effective date: 20180530

REG Reference to a national code

Ref country code: NO

Ref legal event code: T2

Effective date: 20180425

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180725

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180726

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 992982

Country of ref document: AT

Kind code of ref document: T

Effective date: 20180425

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180827

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602013036731

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20190128

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

REG Reference to a national code

Ref country code: CH

Ref legal event code: PCAR

Free format text: NEW ADDRESS: ROUTE DU COUTSET 18, 1485 NUVILLY (CH)

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190306

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180825

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20130306

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180425

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NO

Payment date: 20230309

Year of fee payment: 11

Ref country code: LU

Payment date: 20230227

Year of fee payment: 11

Ref country code: IE

Payment date: 20230110

Year of fee payment: 11

Ref country code: FI

Payment date: 20230315

Year of fee payment: 11

Ref country code: DK

Payment date: 20230314

Year of fee payment: 11

Ref country code: CZ

Payment date: 20230215

Year of fee payment: 11

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: TR

Payment date: 20230303

Year of fee payment: 11

Ref country code: BE

Payment date: 20230216

Year of fee payment: 11

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230505

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20230402

Year of fee payment: 11

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20240320

Year of fee payment: 12

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20240321

Year of fee payment: 12

Ref country code: GB

Payment date: 20240322

Year of fee payment: 12

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20240320

Year of fee payment: 12

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240306